In 4Pi applications, two objective lenses which are disposed opposite each other with respect to a focal plane and whose optical axes are orthogonal to the focal plane must always be aligned in such a way that their foci meet in exactly one point within the focal plane. High-quality and error-free images can only be produced if the two objective lenses are precisely positioned in this manner.
Presently, very few methods and devices of the type mentioned at the outset have been described. In the known methods, one of the two objective lenses is stationary while the other objective lens is manually moved in the x-, y- and z-directions. By convention, x and y refer to the plane orthogonal to the optical axis, and the z direction is defined to be in the direction of the optical axis. In the known methods, an optical path is coupled out of the 4Pi system, and the interferometric configuration of the 4Pi system is made use of. Specifically, the interferometric image is observed, and the movably disposed objective lens is moved in the x- and y-directions until a zero-order image appears.
The known method is problematic in many aspects. First of all, the procedure described can only be used to adjust the two objective lenses in the x- and y-directions, while the adjustment in the z-direction requires a completely different method. In practice, this means that a user has to switch between the two methods during adjustment, which results in a significant loss of time for the user. Another disadvantage is that the state in which the two objective lenses are precisely adjusted with respect to each other does not remain stable over a longer period of time because of drifts or vibrations. As a result of this, image acquisition is possible just as long as the completed adjustment is stable. In the event of a misalignment, the measurement procedure must be terminated, the two objective lenses must be precisely realigned with each other, and it is only then that the measurement can be continued. Not the least is it attributable to this complicated and time-consuming adjustment, which also requires a high level of knowledge and experience, that no real breakthrough has yet been achieved as far as a routine application of 4Pi microscopy is concerned.